All muscle movements are controlled by the output of motor neurons whose cell bodies are located in the spinal cord. Corticospinal, sensory, and inter-neurons form synapses on motor neurons early in embryonic development, establishing neural circuits that enable voluntary muscle movements, reflexes, and locomotor activity. Recent studies demonstrate that the formation of specific circuits depends upon the settling position of motor neurons within the spinal cord topographic map. Though axonal guidance has been extensively studied as a component of neural connectivity, little is known about motor neuron migration and cell adhesion despite their importance in determining cell body position. Furthermore, neuronal migration defects underlie many rare but devastating neurological conditions such as Lissencephaly that end in childhood death. The combinatorial expression of transcription factors can be used to identify motor neurons based upon subtype identity. By performing immunohistochemistry using transcription factor antibody markers, the position of motor neuron subtypes was identified in spinal cords of wild type and Robo1-/-2-/- (DKO) mice. Preliminary results demonstrate multiple interneuron and motor neuron positioning defects in Robo DKO mice and, additionally, in chicks electroporated with Slit2N-expressing plasmids compared to wild-type. These results demonstrate, for the first time, that the repulsive guidance cue Slit and its receptor Robo influence motor neuron and interneuron settling position during critical stages of motor circuit development. In-situ hybridization data identifies Slit2 and Robo co-expression by motor neurons, and it is hypothesized that these molecules are engaged in a novel cell-autonomous signaling pathway that potentially silences motor neurons from a Slit2 repulsive gradient established by the floor plate. Conditional knockout mice will be used to genetically ablate Slit2 expression in motor neurons and in the floor plate, and defects in motor neuron position will be identified to determine if cell-autonomous signaling is occurring (Aim1). Since Slit and Robo are known mediators of cell adhesion and adhesion molecules regulate motor neuron subtype clustering, it is hypothesized that mis-positioning defects in Robo DKO mice are due to disrupted cell-cell adhesion. This possibility will be investigated by performing an adhesion molecule screen to visualize changes in their expression and localization in motor neurons (Aim2). Finally, preliminary results demonstrate motor column- specific expression patterns of Robo isoforms and motor column-specific mis-positioning defects in Robo DKO mutants. From these findings, it is hypothesized that Robo isoforms mediate motor column-specific actions. By studying neuronal mis-positioning defects in Robo1-/- and Robo2-/- single knockout mice, it will be determined if Robo isoforms have differential signaling capacities (Aim3). Taken together, these proposed studies will help uncover fundamental mechanisms by which Slit and Robo are able to specify motor neuron settling position, a critical determinant of synaptic connectivity in the spinal cord.